High coverage stlfr

Abstract

Described herein are high coverage single tube Long Fragment Read (stLFR) technology which uses performs stLFR on target DNA fragments that have already been amplified before they are co-barcoded, which provides higher amount of DNA for sequencing and increases sequencing coverage. In some embodiments, the high coverage stLFR described in this application uses two rounds of stLFR. In some embodiments, the target DNA fragments are transposed with transposons having particular positional barcodes that can be used to order sequence reads.

1 . A method for preparing a library of barcoded polynucleotides for determining a sequence of a target nucleic acid(s) comprising
 (a) providing fragments derived from the target nucleic acid, wherein the fragments are double-stranded or partially double-stranded;   
       (b) introducing staggered single-stranded breaks into at least some double-stranded fragments, thereby producing a plurality of first complexes, wherein each first complex comprises a plurality of first subfragments, and 
       (c) associating first capture oligonucleotide sequences with at least some of the first subfragments,
 wherein each first capture oligonucleotide sequence comprises a first barcode, and optionally comprises a promoter sequence or a primer binding sequence, and 
 wherein the associating comprises combining the double-stranded fragments in (a) or the complexes in (b) with a plurality of individual first beads, wherein each individual first bead comprises a plurality of first capture oligonucleotides immobilized thereon, wherein each capture oligonucleotides comprises a first capture oligonucleotide sequence, wherein the first capture oligonucleotides immobilized on each individual first bead comprises the same first capture oligonucleotide sequence, wherein a majority of different first beads have different first capture oligonucleotides immobilized thereon, and wherein each different first capture oligonucleotide sequence comprises a different first barcode, 
 thereby providing barcoded first subfragments; 
 (d) amplifying at least a portion of the barcoded first subfragments to produce amplified barcoded first subfragments, wherein the amplified barcoded first subfragments are double-stranded or partially double-stranded; 
 (e) introducing staggered single-strand breaks into some of the amplified barcoded first subfragments to generate second complexes, each comprising a plurality of second subfragments; and 
 (f) associating second capture oligonucleotide sequences with at least some of the second subfragments; 
 wherein the associating comprises combining the amplified barcoded first subfragments in (d) or the second complexes in (e) with a plurality of individual second beads, wherein each individual second bead comprises a plurality of second capture oligonucleotides immobilized thereon, wherein each second capture oligonucleotide comprises a second capture oligonucleotide sequence, wherein the second capture oligonucleotides immobilized on each individual second bead comprises the same second capture oligonucleotide sequence, wherein a majority of different second beads have different second capture oligonucleotides immobilized thereon, and wherein each different second capture oligonucleotide sequence comprises a different second barcode, 
 thereby providing a library of barcoded second subfragments. 
 
     
     
         2 . The method of  claim 1 , wherein the average length of the first subfragments is at least 2×greater than the average length of second subfragments in size. 
     
     
         3 . The method of  claim 1 , wherein step (c) is performed in a single mixture, wherein the number of first beads is greater than the number of target nucleic acid fragments in the single mixture, and wherein each first bead comprises multiple copies of the first capture oligo, immobilized thereon. 
     
     
         4 . The method of  claim 3 , wherein first insertion oligonucleotides are added by ligation or by synthesis to at least some first subfragments in step (b), and wherein step (c) further comprises:
 (1) ligating the first capture oligonucleotides to the first insertion oligonucleotides, or   (2) hybridizing first capture oligonucleotides to the first insertion oligonucleotides and then extending the insertion oligonucleotides by a DNA polymerase to incorporate first barcodes.   
     
     
         5 . The method of  claim 1 , wherein step (f) is performed in a single mixture and wherein the number of second beads is greater than the number of the amplified barcoded first subfragments in the single mixture, wherein each second bead comprises multiple copies of the second capture oligo, immobilized thereon. 
     
     
         6 . The method of  claim 5 , wherein each of at least some second subfragments is linked to a second insertion oligonucleotide, and wherein step (e) further comprises:
 (1) ligating the second capture oligonucleotide to the second insertion oligonucleotide, or   (2) hybridizing the second capture oligonucleotide to the second insertion oligonucleotide and then extending the insertion oligonucleotide by a DNA polymerase to incorporate second barcodes.   
     
     
         7 - 10 . (canceled) 
     
     
         11 . The method of  claim 4 , wherein the first insertion oligonucleotide is hybridized to a complementary oligo to form a partially double-stranded first insertion oligonucleotide, and Wherein the first insertion oligonucleotide is ligated to at least some of the breaks by 3′ branch ligation. 
     
     
         12 - 25 . (canceled) 
     
     
         26 . The method of  claim 4 , wherein each of the first insertion oligonucleotides comprises a first positional barcode, wherein different first insertion oligonucleotides comprises different first positional barcodes, and/or
 wherein each of the second insertion oligonucleotides comprises a second positional barcode, wherein different second insertion oligonucleotides comprises different second positional barcodes.   
     
     
         27 . (canceled) 
     
     
         28 . A method of analyzing the full-length sequence of one or more target region comprising
 (a) amplifying each target region,   (b) ligating an adaptor oligonucleotide to both ends of amplified nucleic acid target fragments comprising the target region,   (c) introducing staggering single-stranded breaks in at least some of the amplified nuceic acid fragments from (b), to produce a plurality of first complexes each comprising a plurality of first subfragments,   (d) introducing first capture oligonucleotides to at least some of the first subfragments, wherein each first capture oligonucleotides comprises
 (1) optionally a promoter sequence or a primer binding sequence, and 
 (2) a first barcode, wherein first capture oligonucleotides immobilized on the same individual bead comprise the same first barcode, and a majority of beads have different first barcodes, thereby providing barcoded first subfragments. 
   
     
     
         29 - 38 . (canceled) 
     
     
         39 . The method of  claim 4 , wherein at least some of the first insertion oligonucleotides each comprises a positional barcode, wherein different first insertion oligonucleotides comprise different positional barcodes. 
     
     
         40 . The method of  claim 6 , wherein at least some of the second insertion oligonucleotides each comprises a positional barcode, wherein different second insertion oligonucleotides comprise different positional barcodes. 
     
     
         41 - 43 . (canceled) 
     
     
         44 . A method of inserting oligonucleotides into fragments of a target nucleic acid comprising:
 (a) introducing staggered single-stranded breaks into the fragments,   (b) contacting the fragments from (a) with an insertion scaffold, wherein the adaptors are anchored to the scaffold and separated by predetermined spacing, wherein the insertion scaffold comprises a plurality of double-stranded or partially double-stranded adaptors and a scaffold,   wherein each adaptor comprises an insertion oligonucleotide comprising a unique positional barcode, and   wherein the contacting results in the plurality of insertion oligonucleotides being introduced into the fragments at the single-stranded breaks, thereby producing first insertion complexes, each comprising a plurality of first subfragments.   
     
     
         45 . The method of  claim 44 , wherein the method further comprises:
 dissociating the scaffold from the plurality of adaptors that have been inserted into the target nucleic acid.   
     
     
         46 . The method of  claim 44 , wherein the method comprises
 contacting a plurality of scaffolds with each of some of the nucleic acid fragments, wherein adaptors in different scaffolds have different scaffold barcodes.   
     
     
         47 . The method of  claim 44 ,
 wherein the scaffold is a single-stranded nucleic acid molecule, and   wherein each adaptor further comprises
 a scaffold hybridization sequence, 
   wherein the transposon hybridizes to the scaffold via the scaffold hybridization sequence, and   wherein the scaffold hybridization sequence can be cleaved to dissociate the scaffold from the adaptor,   wherein the insertion oligonucleotide further comprises a scaffold barcode shared by all adaptors within the scaffold.   
     
     
         48 . The method of  claim 44 , wherein the method further comprise:
 (c) combining in a single mixture (i) the first insertion complexes produced from (a); and (ii) a population of first beads, wherein each first bead comprises multiple copies of a first capture oligonucleotides immobilized hereon, said first capture oligonucleotides comprising a first barcode, wherein the first capture oligonucleotides immobilized on the same individual first bead comprise the same first barcode and a majority of beads have different first barcodes,   (d) for each of a plurality of the first subfragments, introducing the first capture oligonucleotide, thereby producing barcoded first subfragments each is linked to a copy of the first barcode.   
     
     
         49 . The method of  claim 40 , wherein the method further comprise:
 (e) amplifying the plurality of barcoded first subfragments,   (f) introducing staggered single-stranded breaks to the amplified barcoded subfragments, and   (g) contacting with the products from step (f) with second insertion scaffolds, wherein the second insertion scaffolds each comprises a plurality of second adaptors anchored to a second insertion scaffold, thereby introducing second insertion oligonucleotides on the second adaptors to the amplified barcoded first subfragments to produce second insertion complexes, each comprising a plurality of second subfragments.   
     
     
         50 . The method of  claim 49 , wherein the scaffold of the second insertion scaffold is a single-stranded nucleic acid molecule, and each of the second adaptor comprises:
 (1) a second scaffold hybridization sequence,   wherein the second adaptor hybridizes to the scaffold via the second scaffold hybridization sequence, and   wherein the second insertion scaffold hybridization sequence can be cleaved to dissociate the scaffold from the second adaptor;   (2) a second insertion oligonucleotide comprising a unique positional barcode, and   
       a second scaffold barcode shared by the second adaptors within the scaffold of the second insertion scaffold. 
     
     
         51 . The method of claim  41 , wherein the second insertion complexes are mixed with a population of second beads, wherein each bead comprises second capture oligonucleotides immobilized hereon, said oligo nucleotides comprising a second barcode, wherein the oligonucleotides immobilized on the same individual bead comprise the same second barcode and a majority of beads have different second barcodes,
 (g) for each of at least some of the second insertion complexes, introducing multiple copies of the second capture oligonucleotide comprising a second barcode, wherein the multiple copies are from a single bead, and thereby producing a plurality of barcoded second subfragments, each is linked to at least one copy of the second barcode.   
     
     
         52 . The method of  claim 44 , wherein the method further comprises:
 sequencing the plurality of second subfragments to produce a number of sequencing reads.   
     
     
         53 - 56  (canceled) 
     
     
         57 . A plurality of insertion scaffolds,
 wherein each of a plurality of insertion scaffolds comprises   (1) a plurality of adaptors, wherein the adaptors are double-stranded or partially double-stranded, and   (2) a scaffold, and the adaptors are anchored to the scaffold and separated by predetermined spacing,   wherein for each insertion scaffold, each adaptor in the insertion scaffold carries a unique positional barcode and a common scaffold barcode, and   wherein adaptors in different insertion scaffolds have different scaffold barcodes.   
     
     
         58 - 59 . (Canceled) 
     
     
         60 . A nucleic acid complex comprising a plurality of insertion scaffolds of  claim 57 , and a nuclear acid fragment, wherein the plurality of insertion scaffolds are hybridized to the target nuclear acid fragment. 
     
     
         61 . A reaction mixture that is in a single vessel, wherein the reaction mixture comprises a plurality of insertion scaffolds of  claim 57  and multiple fragments derived from a target nucleic acid.